University of Oulu

Li, T.; Tynkkynen, T.; Ihanus, A.; Zhao, S.; Mäkinen, V.-P.; Ala-Korpela, M. Characteristics of Normalization Methods in Quantitative Urinary Metabolomics—Implications for Epidemiological Applications and Interpretations. Biomolecules 2022, 12, 903.

Characteristics of mormalization methods in quantitative urinary metabolomics : implications for epidemiological applications and interpretations

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Author: Li, Tianqi1,2,3; Tynkkynen, Tuulia1,4; Ihanus, Andrei1,2,4;
Organizations: 1Systems Epidemiology, Faculty of Medicine, University of Oulu, 90014 Oulu, Finland
2Center for Life Course Health Research, Faculty of Medicine, University of Oulu, 90014 Oulu, Finland
3Biocenter Oulu, University of Oulu, 90014 Oulu, Finland
4NMR Metabolomics Laboratory, School of Pharmacy, University of Eastern Finland, 70210 Kuopio, Finland
5Computational and Systems Biology Program, Precision Medicine Theme, South Australian Health and Medical Research Institute, Adelaide, SA 5000, Australia
6Australian Centre for Precision Health, Unit of Clinical and Health Sciences, University of South Australia, Adelaide, SA 5000, Australia
Format: article
Version: published version
Access: open
Online Access: PDF Full Text (PDF, 5.3 MB)
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Language: English
Published: Multidisciplinary Digital Publishing Institute, 2022
Publish Date: 2023-05-29


A systematic comparison is presented for the effects of seven different normalization schemes in quantitative urinary metabolomics. Morning spot urine samples were analyzed with nuclear magnetic resonance (NMR) spectroscopy from a population-based group of 994 individuals. Forty-four metabolites were quantified and the metabolite–metabolite associations and the associations of metabolite concentrations with two representative clinical measures, body mass index and mean arterial pressure, were analyzed. Distinct differences were observed when comparing the effects of normalization for the intra-urine metabolite associations with those for the clinical associations. The metabolite–metabolite associations show quite complex patterns of similarities and dissimilarities between the different normalization methods, while the epidemiological association patterns are consistent, leading to the same overall biological interpretations. The results indicate that, in general, the normalization method appears to have only minor influences on standard epidemiological regression analyses with clinical/physiological measures. Multimetabolite normalization schemes showed consistent results with the customary creatinine reference. Nevertheless, interpretations of intra-urine metabolite associations and nuanced understanding of the epidemiological associations call for comparisons with different normalizations and accounting for the physiology, metabolism and kidney function related to the normalization schemes.

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Series: Biomolecules
ISSN: 2218-273X
ISSN-E: 2218-273X
ISSN-L: 2218-273X
Volume: 12
Issue: 7
Article number: 903
DOI: 10.3390/biom12070903
Type of Publication: A1 Journal article – refereed
Field of Science: 1182 Biochemistry, cell and molecular biology
Funding: M.A.-K. was funded by a research grant (number 1416) from the Sigrid Juselius Foundation.
Dataset Reference: The NFBC data used in the current study are available through an application process for researchers who meet the criteria to access confidential data:, accessed on 30 August 2021.
Copyright information: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (